Hormone and growth factor phosphoglycokine mimetics from mycobacterium

ABSTRACT

A hormone or growth factor mimetic second messenger is derived from a microorganism of the genus Mycobacterium, suitably M. vaccae. The mimetic second messenger may mimic the action of insulin, ACTH, NGF, EGF, FGF, TGFβ or HGF. Further, methods of treating type I or type II diabetes mellitus, polycystic ovary syndrome, central nervous system damage, hepatic damage, alcohol abuse, drug sensitivity, tissue damage, adrenal atrophy, etc., are also disclosed. The methods are carried out by administering the mimetic second messenger to a patient in need thereof.

BACKGROUND

1. Field of the Invention

The present invention relates to second messengers which mimic theaction of insulin and other mammalian growth factors and hormones.

2. Related Art

Non insulin-dependent diabetes mellitus is one of the most commonmetabolic disorders in the industrial world. Associated with thedisorder are dyslipidemias, atherosclerosis, hypertension,cardiovascular disorders and renal dysfunction. Obesity constitutes thegreatest risk factor for the disease. Two physiological defects thatlead to the development of diabetes are tissue resistance to the effectsof insulin and altered secretion of insulin.

In order for new treatments of this disorder to be developed it isnecessary to understand the specifics of the insulin signalling pathwaysand other signalling pathways which may interfere with insulin action.It has recently been demonstrated that low molecular weightphosphorylated inositolglycans (IPGs), are released upon insulinstimulation in a tissue-specific manner. These compounds are in thefamily of phosphoglycokines (PGK), defined as biologically active lowmolecular weight compounds containing phosphorylated carbohydrates. Thetissue-derived IPGs mediate some of the actions of insulin. Suchinsulin-mimetics have therapeutic potential in that they could:

(i) substitute for insulin either as a parenteral or oral treatment inpatients with diabetes where the primary pathology relates either todecreased synthesis (type I diabetes) or lack of bioavailable insulin(defects in conversion of proinsulin to insulin or in the formation ofanti-insulin antibodies).

(ii) be used to treat patients with tissue insulin resistance, which isseen in many cases of adult onset or type II diabetes.

(iii) be used to treat or prevent complications of diabetes includingdyslipidemias, atherosclerosis, hypertension, cardiovascular disordersand renal dysfunction. It has further been found that the IPGs are ableto cross the blood brain barrier and affect cerebral glucose and energymetabolism. Since insulin itself has limited ability to cross the bloodbrain barrier, release of the compounds into the circulation followinginsulin stimulation may be crucial in the control of energy metabolismin the brain. In clinical trials, tissue-derived IPGs have been shown tobe effective in reversing age-associated memory loss and in providing aprotective effect under cerebral hypoxic conditions.

As detailed below, the inositolphosphoglycan second messenger signaltransduction effect has also been shown to be functionally relevant forthe signalling of other growth factors, including fibroblast growthfactor (important in wound healing), transforming growth factor β(important in autoimmunity) and hepatocyte growth factor (also known asscatter factor), that together with other growth factors, is importantfor the regeneration of liver tissue following damage by infection,alcohol abuse, drug sensitivity, or autoimmunity.

SUMMARY OF THE INVENTION

The present invention provides a valuable source of phosphoglycokines(PGK) which mimic the activity of the tissue-derived IPGs, which areotherwise not readily available. Only very small quantities of the IPGscan be isolated from mammalian tissues. Since the IPGs are non-proteinin composition, they cannot be produced by recombinant DNA technology.Synthetic chemistry approaches are complicated by the current lack ofstructural details of the tissue-derived IPGs and the complicationsassociated with oligosaccharide syntheses.

Immunotherapy with M. Vaccae

We have previously described the use of antigenic and/or immunoregulatory material derived from Mycobacterium vaccae in the treatmentof tuberculosis (see, for example, British Patent No. 2156673 and U.S.Pat. No. 4,724,144). In our International Patent Application No.PCT/GB90/01169 (publication No. WO91/01751), we have described the useof the same material for immunoprophylactic treatment against AIDS, i.e.for increasing the period between infection by HIV and development ofAIDS.

Mycobacterium vaccae has also been shown to have therapeutic potentialas a treatment for patients infected both with Human ImmunodeficiencyVirus (HIV) and tuberculosis as described by Stanford, J. L. in AIDS(1993) 7, pp 1275-1277. The mechanism of the immunotherapeutic effect isnot fully established, but may relate to the ability of compounds withinthe organism to evoke a Th1 pattern of T cell response to proteins richin epitopes shared between mycobacterial species as described by Boyden,S. V. in J. Immunol. (1955), 75, pp 15. Human homologues of several ofthese proteins are implicated in human autoimmune diseases such asrheumatoid arthritis and perhaps also in schizophrenia, and M. vaccaemay also have relevant immunoregulatory properties in these conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing both the liver A and P-type IPGs were ableto stimulate proliferation of the fibroblasts in serum free medium.

FIG. 2 is a bar graph showing various dilutions of M. vaccae derived PGKsecond messengers co-purify with liver derived A-type IPGs.

FIG. 3 is a bar graph showing various dilutions of M. vaccae derived PGKsecond messengers co-purify with liver derived A-type IPGs.

FIG. 4 is a bar graph showing various dilutions of M. vaccae derived PGKsecond messengers which co-purify with liver derived P-type IPGs.

FIG. 5 is a bar graph showing various dilutions of M. vaccae derived PGKsecond messengers co-purify with liver derived P-type IPGs.

FIG. 6A is a bar graph showing the effects of low and high VSB-Aconcentrations on cell proliferation; culture medium (RPMI only),culture medium plus 7% FCS, or medium plus various dilutions of M.vaccae derived PGK second messengers which co-purify with liver derivedA-type IPGs.

FIG. 6B is a bar graph comparing the effects of low and high VBS-Aconcentrations on cell proliferation.

FIG. 7A is a bar graph showing VSB-P inhibited cell proliferationsecondary to stimulation of steroid production and

FIG. 7B is a bar graph showing VBS-P inhibited cell proliferationsecondary to stimulation of steroid production.

DETAILED DESCRIPTION OF THE INVENTION

We have unexpectedly found that phosphoglycokines (PGK) which co-purifywith insulin-mimetic inositolphosphoglycans (IPG) from rat or humanliver can be obtained from cultures of Mycobacterium vaccae. The M.vaccae derived products are able to mimic the action of mammalian IPGsecond messengers in the following ways:

(i) stimulation of EGF(Rc) transfected 3T3 cells,

(ii) stimulation of pyruvate dehydrogenase phosphatase activity,

(iii) inhibition of cAMP dependent protein kinase activity, and

(iv) stimulation of lipogenesis in isolated adipocytes.

It is also probable that the M. vaccae derived products modulate steroidmetabolism in adrenal cells.

It is clear that M. vaccae and related strains of mycobacteria are asource of PGKs which either mimic the activity of, or are very similarin structure to, the IPG type of PGK second messengers present inmammalian tissues. Previously, such second messengers could only beisolated in extremely small quantities from mammalian tissues, such asliver. We have surprisingly found that compounds extracted from M.vaccae mimic the action of the IPG second messengers. This providesadvantages over material derived from liver tissue, in both ease ofextraction and in the quantities of messenger which may be obtained.

The present invention accordingly provides a hormone and growth factormimetic second messenger derived from a mycobacterium, preferably fromMycobacterium vaccae. The mimetic second messenger may mimic the actionof a number of hormones and growth factors. For example, the mimeticsecond messenger may mimic the action of insulin, adrenocorticotropichormone (ACTH), nerve growth factor (NGF), epidermal growth factor(EGF), fibroblast growth factor (FGF), transforming growth factor β(TGFβ), and hepatocyte growth factor (HGF).

The mimetic second messenger is a low molecular weight phosphoglycokinethat is or that mimics a phosphorylated inositolglycan (IPG) ofmammalian origin.

The invention further provides a use of an insulin mimetic secondmessenger derived from a mycobacterium, preferably Mycobacterium vaccae,in the preparation of a medicament for the treatment of Type I or TypeII diabetes mellitus, polycystic ovary syndrome, lipodystrophy,age-related memory loss, and post-ischaemic damage secondary to strokeor post-transplant complications.

The invention further provides a use of a nerve or neurite growth factormimetic second messenger derived from a mycobacterium, preferablyMycobacterium vaccae, in the preparation of a medicament for thetreatment of nerve, spinal cord or central nervous system damagesecondary to trauma, or autoimmune or metabolic damage, orpost-ischaemic damage secondary to stroke or post-transplantcomplications.

The invention also provides a use of a hepatocyte growth factor mimeticsecond messenger derived from a mycobacterium, preferably Mycobacteriumvaccae, in the preparation of a medicament for the treatment of hepaticdamage caused by infection, alcohol abuse, drug sensitivity, orautoimmunity.

The invention also provides a use of a fibroblast growth factor mimeticsecond messenger and an epidermal growth factor mimetic second messengerderived from a mycobacterium, preferably Mycobacterium vaccae, in thepreparation of a medicament for the promotion of wound healing followingsurgery or trauma or tissue damage induced by ischaemia or autoimmunity.

The invention also provides a use of an adrenal cell growth factormimetic second messenger and an ACTH mimetic second messenger derivedfrom a mycobacterium, preferably Mycobacterium vaccae, in thepreparation of a medicament for the treatment of disease statesinvolving adrenal atrophy such as tuberculosis.

The invention further provides a pharmaceutical composition comprising ahormone or growth factor mimetic second messenger as defined herein.

The invention also provides methods of treatment of

(i) type I or type II diabetes mellitus, polycystic ovary syndrome,lipodystrophy, age-related memory loss and post-ischemic damagesecondary to stroke or post-transplant complications, which comprisesadministering an insulin mimetic second messenger derived from amicroorganism of the genus Mycobacterium, preferably M. vaccae;

(ii) nerve, spinal chord or central nervous system damage secondary totrauma, autoimmune or metabolic damage, or post-ischaemic damagesecondary to stroke or post-transplant complications which comprisesadministering a nerve or neurite growth factor mimetic second messengerderived from a microorganism of the genus Mycobacterium, preferably M.vaccae;

(iii) hepatic damage caused by infection, alcohol abuse, drugsensitivity or autoimmunity which comprises administering a hepatocytegrowth factor mimetic second messenger derived from a microorganism ofthe genus Mycobacterium, preferably M. vaccae;

(iv) a disease state involving adrenal atrophy, such as tuberculosis,which comprises administering an adrenal cell growth factor mimeticsecond messenger and an ACTH mimetic second messenger derived from amicroorganism of the genus Mycobacterium, preferably M. vaccae.

The invention also further provides

(v) a method for the promotion of wound healing following surgery ortrauma or tissue damage induced by ischaemia or autoimmunity whichcomprises administering a fibroblast growth factor mimetic secondmessenger and an epidermal growth factor mimetic second messengerderived from a microorganism of the genus Mycobacterium, preferably M.vaccae.

Cell Signalling

A number of examples of cell-signalling arrangements have been describedin the literature. At least three classes of cell surface receptors areinvolved in cellular regulation. A single transmembrane spanning domainand multiple membrane-spanning domains are described by Lowe, D. G. inEMBO (1989) 8, 1377-1384. Domains with GPI membrane anchors aredescribed by Bamezai and Rock in Oncogene (1991) 6, 1445-1451.

The receptor tyrosine kinases (TRK), including the insulin receptorrequire ligand-stimulated kinase activity for a biological response,according to Lammers in EMBO (1989) 8, 1369-1375. Protein-proteininteractions which occur beyond kinase activation have been described indetail for a number of growth factor specific receptors. These canbroadly be classified into pathways which result in the translocation ofactivated protein kinases into the nucleus where they phosphorylate andactivate nuclear transcription factors such as described by Egan andWeinberg in Nature (1993) 365, 781-783, or those which involvephosphorylation and activation of transcription factor subunits in thecytoplasm which then translocate to the nucleus and induce transcriptionas described by e.g. Muller in Nature (1993) 366, 129-135.

i) Inositolphosphoglycan second messengers are released from the celland are active when added extracellularly

None of the currently described signalling pathways can explain thecommunity effect whereby a critical density of cells is required beforea biological response can be supported. This common biologicalphenomenon suggests the existence of an extracellular loop involved incell signalling. It has previously been reported that, upon growthfactor stimulation, low molecular weight non-peptide factors arereleased into the medium. These factors are then able to mimic some ofthe actions of that growth factor when added to unstimulated cells.These can therefore be regarded as "second messengers". Preliminarystructural analysis has suggested that these compounds contain inositol,carbohydrates and phosphate groups, and these compounds have recentlybeen classified as A or P-type inositolphosphoglycans (as definedbelow). These compounds are in the family of phosphoglycokines (PGK),defined as biologically active low molecular weight compounds containingphosphorylated carbohydrates. It has been shown for example byRademacher et al in Brazilian J. Med. Biol. Res. (1994) 27, 327-341,that the precursor forms of the IPGs are glycosylphosphatidylinositols(GPIs).

ii) Integration of growth factor and soluble mediator dependentsignalling Pathways

Many cytokines and growth factors share common signal transductionpathways. It has been proposed that the specificity for each factorcould be achieved through unique tyrosine-phosphorylated proteinstriggered by individual factors. Alternatively a number of accessorysignalling pathways have also been described which give rise to a numberof soluble mediators such as cAMP, IP3, Ca+², cGMP, diacylglcerol andcADPR. Growth factor and soluble mediator-dependent signalling pathwaysmay converge to synergistically stimulate gene expression (e.g. FGF andcAMP). It has recently been suggested by Tan et al in Mol. Cell Biol.(1994) 14, 7546-7556 that, in addition to the IPGs, cADPR is alsoreleased extracellularly. In the cases of both IPGs and cADPR, it is notyet known how they reach their intracellular targets.

iii) Inositolphosphoglycans are involved in the action of many differentgrowth factors and hormones

Inositolphosphoglycan second messengers (IPGs) are able to mimic theaction of a large number of insulin-dependent biological effects such asplacental steroidogenesis, insulin stimulation of adipocytes,hepatocytes, myocytes and T-lymphocytes, and insulin dependentprogesterone synthesis in swine ovary granulosa cells.

In addition, a number of other growth factors also appear to stimulatethe production of IPGs including:

transforming growth factor β,

nerve growth factor,

hepatocyte growth factor,

insulin-like growth factor I (IGF-1),

IgE-dependent activation of mast cells,

ACTH signalling of adrenocortical cells,

activation of human platelets,

FSH and HCG stimulation of granulosa cells,

thyrotropin stimulation of thyroid cells,

cell proliferation in the early developing ear,

rat mammary gland,

control of human fibroblast proliferation, and

IL-2 stimulation of T and B-lymphocytes.

iv) A and P-type mediators related to the action of insulin

The family of myo-inositol-containing IPGs (A-type) has the followingproperties or activities

1) stimulation of lipogenesis in adipocytes,

2) inhibition of cAMP-dependent protein kinase and modification of theactivity of adenylate cyclase and cAMP-phosphodiesterases in order toregulate the level of cAMP in cells, thus contributing to the control ofcAMP and cAMP-regulated intracellular processes, and

3) support in the growth of neurons from the chick embryo statoacousticganglia.

The family of chiro-inositol-containing IPGs (P-type) has the followingproperties or activities:

1) activation of pyruvate dehydrogenase phosphatase (PDH P'ase),glycogen synthase and other enzymes, and

2) support in the growth and differentiation (neurite outgrowth) of theneurons present in the chick statoacoustic ganglion neurons.

Both the A and P type mediators can also support the growth andproliferation of EGF(Rc) transfected NIH 3T3 cells.

v) Role of mediators in insulin signalling and type II diabetes

These compounds are important in insulin signalling. Experiments haveshown that addition of antibody with anti-IPG specificity is able toblock both the metabolic and mitogenic actions of insulin. Furthermore,mutant cells which are unable to synthesize IPGs respond to insulin asdetermined by tyrosine phosphorylation, but are not stimulated to elicitthe metabolic effects of the hormone.

These compounds are also important in the pathogenesis ofinsulin-resistant type II diabetes. It has been recognised that diabeticGK rats have a defect in the synthesis or release of functional IPGs andthat decreased urinary secretion rate of chiro-inositol is directlyassociated with insulin resistance in both human patients with type IIdiabetes and spontaneously diabetic rhesus monkeys. Furthermore,infusion of chiro-inositol into normal rats given a glucose load orstreptozotocin-treated rats results in decreased plasma glucose andenhanced activity of glycogen synthase I.

The preferred mycobacterium is a strain of M. vaccae, most preferablythat denoted by R877R isolated from mud samples from the Lango districtof Central Uganda (J. L. Stanford and R. C. Paul, Ann. Soc. Belge Med,Trop. 1973, 53, 141-389). The strain is a stable rough variant andbelongs to the aurum sub-species. It can be identified as belonging toM. vaccae by biochemical and antigenic criteria (R. Bonicke, S. E.Juhasz., Zentr albl. Bakteriol. Parasitenkd. Infection skr. Hyg. Abt. 1,Orig., 1964, 192, 133).

The strain denoted R877R has been deposited under the terms of Budapestat the National Collection of Type Cultures (NCTC) Central Public HealthLaboratory, Colindale Avenue, London NW9 5HT, United Kingdom on Feb.13th, 1984 under the number NCTC 11659.

The following Figures are included:

FIG. 1. EGF(Rc) transfected 3T3 cells were incubated with culture medium(control), medium plus FCS or medium plus various dilutions of liver Aand P-type IPGs. Both the A and P-type IPGs were able to stimulateproliferation of the fibroblasts in serum free medium. See notes toTable 1 below for the wet weight of tissue to which these dilutionscorrespond.

FIG. 2. EGF(Rc) transfected 3T3 cells were incubated with culture medium(control), medium plus FCS or medium plus various dilutions of M. vaccaederived PGK second messengers which co-purify with liver derived A-typeIPGs. The M. vaccae derived PGK (VSB-A) at a dilution of 1/160 was aspotent as a 1/40 dilution of rat liver derived IPG. See notes to Table 1below for the wet weight of tissue to which these dilutions correspond.

FIG. 3. EGF(Rc) transfected 3T3 cells were incubated with culture medium(control), medium plus FCS or medium plus various dilutions of M. vaccaederived PGK second messengers which co-purify with liver derived A-typeIPGs. The M. vaccae derived PGK (VBS-A) at a dilution of 1/60 was aspotent as a 1/40 dilution of rat liver derived IPG. See notes to Table 1below for the wet weight of tissue to which these dilutions correspond.

FIG. 4. EGF(Rc) transfected 3T3 cells were incubated with culture medium(control), medium plus FCS or medium plus various dilutions of M. vaccaederived PGK second messengers which co-purify with liver derived P-typeIPGs. The M. vaccae derived PGK (VBS-P) at a maximal stimulationdilution of 1/80 was not potent as 10% FCS alone. See notes to Table 1below for the wet weight of tissue to which these dilutions correspond.

FIG. 5. EGF(Rc) transfected 3T3 cells were incubated with culture medium(control), medium plus FCS or medium plus various dilutions of M. vaccaederived PGK second messengers which co-purify with liver derived P-typeIPGs. The M. vaccae derived PGK (VSB-P) at a dilution of 1/40 was not aspotent as 10% FCS. See notes to Table 1 below for the wet weight oftissue to which these dilutions correspond.

FIG. 6, A and B. Y1 adrenal cells were incubated with culture medium(RPMI only), culture medium plus 7% FCS, or medium plus variousdilutions of M. vaccae derived PGK second messengers which co-purifywith liver derived A-type IPGs. At high concentrations (1/40) bothpreparations (VSB-A and VBS-A) stimulated some cell proliferation. Atlower concentrations (1/80-1/1280) proliferation was inhibited. Similarpatterns are seen for ACTH stimulation of the Y1 cells where theinhibition of proliferation is accompanied by steroid production.

FIGS. 7A and 7B. Y1 adrenal cells were incubated with culture medium(RPMI only), culture medium plus 7% FCS, or medium plus variousdilutions of M. vaccae derived PGK second messengers which co-purifywith liver derived P-type IPGs. At all concentrations tested, bothpreparations (VSB-P and VBS-P) inhibited cell proliferation secondary tostimulation of steroid production. Cells were viable at allconcentrations tested.

The invention is further illustrated by the following Examples.

EXAMPLES Growth of M. vaccae

Mycobacterium vaccae NCTC11659 was grown by spreading on the surface ofmodified Sauton's medium, solidified with 1.5% agar. The cultures weremaintained at 32° C. for 3 weeks.

Example 1 Isolation of Second Messengers from M. vaccae

The bacterial growth was scraped off the surface of the modifiedSauton's medium with a spatula, and weighed. Bacteria were suspended in50 mM formic acid, containing 1 mM EDTA and 1 mM β-mercaptoethanol (3 mlof buffer per gram of organisms). Then either of the followingprocedures was adopted:

(i) the organisms in buffer were ultrasonically disrupted for 30 mins ina cooled glass container with the wave peak to peak distance set at 8μ.Then the sonicate was boiled for 3 mins and cooled on ice. When cool itwas centrifuged at 29,500 g for 90 mins at 4° C.

(ii) the organisms in buffer were boiled for 3 mins and cooled on ice.When cool the suspension was ultrasonically disrupted for 30 mins in acooled glass container with the wave peak to peak distance set at 8μ.Then the sonicate was centrifuged at 29,500 g for 90 mins at 4° C.

The clear supernatant from either procedure was then recovered andtreated exactly as for extracts of rat or human tissues as describedbelow.

Example 2 Isolation of Second Messengers from Mammalian Tissues and M.vaccae

A. Isolation of IPGs from rat or human tissues following insulinstimulation

Adult male Wistar rats are starved overnight. The rats are thenanaesthetised by injection of Hypnome and 20 min later are injected viathe tail vein with either 0.1 ml saline or 0.1 ml saline solutioncontaining 50 mU of insulin. After 120 seconds the animals aresacrificed by cervical dislocation, and tissues are removed in thefollowing order: liver, heart, adipose tissue, kidney and muscle. Alltissues are immediately freeze-clamped (liquid nitrogen) and storedfrozen at -80° C. The rats are still normoglycemic at the time of tissueremoval.

In order to extract the IPGs released following insulin stimulation, thefrozen tissue is powdered under liquid nitrogen and the tissue placeddirectly into boiling 50 mM formic acid containing 1 mM EDTA, 1 mMβ-mercaptoethanol (3 ml of buffer per gram (wet weight) of tissue), andhomogenised with an Ultra-Turrex for 30 sec and then boiled for 5minutes. The solution is then cooled on ice and centrifuged at 29,500×gfor 90 minutes at 4° C. The supernatant fraction is recovered and 10mg/ml of activated charcoal added for 10 min with stirring at 4° C. Thecharcoal is removed by centrifugation at 29,500×g for 30 min. at 4° C.and the clear supernatant recovered. The solution was then diluted with10 volumes of water and the pH adjusted to 6.0 with 10% NH₄ OH solutionand then gently shaken overnight with AG1X8 (20-50 mesh, formate form)resin (0.3 ml resin/ml solution). The resin is then poured into achromatography column and washed with 2 bed volumes of water followed by2 bed volumes of 1 mM HCl. The column is then eluted with 10 mM HCl (5bed volumes) to obtain P-type IPGs, and then 50 mM HCl (5 bed volumes)to obtain A-type IPGs. Both fractions are adjusted to pH 4.0 with 10%NH₄ OH solution and then dried in a rotary evaporator (37° C.). Thedried material is redissolved with water and then freeze-dried and thisis repeated twice. Material from two rats is normally combined andsubjected to descending paper chromatography (butanol/ethanol/water4:1:1, Whatman 3MM) for 9 hours and the material in fractions -1 to 7 cmfrom the origin is eluted from the paper with water. After evaporationby freeze-drying, the material is dissolved in 200 μl of Hanks solutionand the pH adjusted to 7 with 1 M KOH. For the case of adipose tissue,after powderizing and boiling, the solution is cooled on ice and thesame volume of chloroform is added. The suspension is then stirred for10 min and then is centrifuged. After centrifugation, the chloroformphase is removed and discarded and the aqueous phase treated as in thecase of the other tissues.

B. Isolation of second messengers from M. vaccae

M. vaccae was heat treated and then sonicated or vice versa. The extractwas then placed directly into boiling 500 mM formic acid containing 1 mMEDTA, 1 mM β-mercaptoethanol (3 ml of buffer per gram (wet weight) oftissue), and homogenised with an Ultra-Turrex for 30 sec and then boiledfor 5 minutes. The solution was then cooled on ice and centrifuged at29,500×g for 90 minutes at 4° C. The supernatant fraction was recoveredand 10 mg/ml of activated charcoal added for 10 min with stirring at 4°C. The charcoal was removed by centrifugation at 29,500×g for 30 min at4° C. and the clear supernatant was recovered. The solution was thendiluted with 10 volumes of water and the pH adjusted to 6.0 with 10% NH₄OH solution and was then gently shaken overnight with AG1X8 (formateform) resin (0.3 ml resin/ml solution). The resin was then poured into achromatography column and washed with 2 bed volumes of water followed by2 bed volumes of 1 mM HCl. The column was then eluted with 10 mM HCl (5bed volumes) to obtain PGK eluting under the same conditions asmammalian P-type IPGs and then 50 mM HCl (5 bed volumes) to obtain PGKeluting under the same conditions as mammalian A-type IPGs. Bothfractions were adjusted to pH 4.0 10% NH₄ OH solution and dried in arotary evaporator (37° C.). The dried material was redissolved in waterand was then freeze-dried, this was repeated twice. The extracts werethen subjected to descending paper chromatography (butanol/ethanol/water4:1:1, Whatman 3MM) for 9 hours and the material in fractions -1 to 7 cmfrom the origin were eluted from the paper with water. After evaporationby freeze-drying, the material was dissolved in 200 μl of Hanks solutionand pH was adjusted to 7 with 1 M KOH.

Example 3 In vitro Effects of M. vaccae Derived Second Messengers onPhosphatase and Kinase Activities and Lipogenesis and Comparison withLiver-Derived IPGs

(a) pyruvate dehydrogenase phosphatase assay

The activation is followed spectrophotometrically as described by Lilleyet al. in Arch. Biochem. Biophys. Res. Commun. (1992) 166, 765-771.

(b) cAMP-dependent protein kinase assay

The inhibition of cAMP-PK is measured by following the phosphorylationof histone II by ³² P-ATP.

(c) lipogenesis assay

The activation of lipogenesis is monitored by measuring theincorporation of uniformly labelled glucose into lipids of isolatedadipocytes as described by Rodbell in J. Biol. Chem. (1964) 239,375-380.

Results

Table 1 summarises the action of the M. vaccae derived IPG secondmessengers and compares the qualitative and quantitative pattern ofactivities to that of the rat liver derived IPGs. Two preparations of M.vaccae-derived PGK were used for the experiments, and similar resultswere obtained for both preparations. Table 1 clearly demonstrates thatthe M. vaccae derived PGK second messengers which co-purify with liverderived P-type IPGs are able to inhibit cAMP dependent protein kinase,stimulate pyruvate dehydrogenase phosphatase and stimulate proliferationof EGF(Rc) transfected 3T3 cells. Similarly, the M. vaccae derived PGKsecond messengers which co-purify with the liver derived A-type IPGs areable to inhibit cAMP dependent protein kinase, stimulate lipogenesis ofrat adipocytes and stimulate proliferation of EGF (Rc) 3T3 cells.

Example 4 Stimulation of EGF Receptor Transfected 3T3 Cells by M. vaccaeDerived Second Messengers in Serum Free Medium and Comparison withLiver-Derived IPGs

Stock cells are grown in flasks with DMEM containing 10% FCS plus 100units/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine until thecells approach 80-90% confluence. The cells are released from the plateusing trypsin (0.25%) and 10⁴ cells are added to each well of 96 wellmicrotiter plates in 100 μl medium. The cells are allowed to adhere for24 hours in full medium. The medium is then removed and cells washedtwice with 100 μl of Hanks solution. The cells are then incubated inDMEM without FCS for 24 hours. After 24 hours the medium is removed andDMEM containing no FCS, plus FCS, or PGK alone is added. After 18 hours,³ H-thymidine is added per well and incubation continued for 4-6 hours.The medium is then removed, cells washed and harvested followingtrypsinization. Incorporation of radioactive ³ H-thymidine into DNA isdetermined by transferring cell suspensions to Whatman GF/C filter disksusing a cell harvester. Radioactivity is measured by scintillationcounting.

Results

FIG. 1 shows the response of EGF(Rc) transfected 3T3 fibroblasts to ratliver derived A and P-type IPGs. Both mediators at maximal concentrationare more potent than 10% FCS in stimulating cell proliferation. FIGS. 2and 3 show the effect of the M. vaccae derived PGK second messengerswhich co-purify with liver A-type IPG on cell proliferation. LiverA-type IPG showed stimulation greater than that for FCS alone. Twoseparate preparations of M. vaccae-derived PGK gave similar results.FIGS. 4 and 5 show the effect of the M. vaccae derived second messengerswhich co-purify with liver P-type IPG on cell proliferation. While bothM. vaccae preparations were able to stimulate cell proliferation theywere not as effective as FCS alone. These results suggest that M. vaccaepredominantly releases PGKs that mimic A-type second messengers, andreleases lesser amounts of PGKs that mimic P-type second messengers.This pattern is found for IPG release in adipose and heart tissuefollowing insulin stimulation (data not shown), in contrast to kidneyand liver which release equal amounts of A and P-type mediators.

Example 5 Effect of M. vaccae Derived Second Messengers on Proliferationof Y1 Adrenal Cells in Serum Free Medium

The Y1 cell line is derived from a murine adrenal carcinoma andexpresses many of the enzymes involved in steroid biosynthesis, as wellas functional adrenocorticotropin (ACTH) receptors. There is evidencethat adrenal cells contain inositol phosphoglycans and that ACTHstimulates breakdown. This is followed by synthesis ofphosphatidylinositolglycans in these cells. Thus it is likely thatinositolphosphoglycans can act as second messengers for this receptor.The line is maintained in RPMI 1640 tissue culture medium, supplementedwith glutamine (2 mM) and 7% fetal calf serum. The cells adhere to theplastic, and before growth becomes confluent (2-4 days), the cells areharvested using 0.02% w/v EDTA and trypsin (0.025%) inphosphate-buffered saline, washed, resuspended in complete tissueculture medium and divided between 2 or 3 tissue culture flasks. Forassay of PGK extracts of mycobacteria (whether of the type that eluteunder the same conditions as A-type or P-type mammalian IPG), cells areharvested as described above and are then plated into the wells of 96well microtiter tissue culture plates, 10⁴ cells in 100 μl of RPMI 1640plus glutamine and FCS. After incubation for 24 hours to allowattachment of the cells to the plate, the wells are washed thoroughlywith unsupplemented RPMI 1640 plus glutamine only. The medium iswithdrawn and replaced with:

(i) RPMI 1640 with glutamine but no serum or serum substitute (negativecontrol)

(ii) RPMI 1640 with glutamine and 7% FCS (positive control)

(iii) RPMI 1640 with glutamine and final dilutions of M. vaccae-derivedPGK second messengers (for example from 1/40 to 1/320 dilution of stocksolution).

After incubation for a further 18-24 hours, 0.2 μCi of ³ H-thymidine isadded to each well in unsupplemented RPMI 1640. Incubation is continuedfor 8-16 hours and then the medium is withdrawn, cells are released fromthe plastic with EDTA/trypsin as described above, and are harvested fordetermination of incorporation of ³ H thymidine into DNA by liquidscintillation counting, according to standard protocols.

Results

Y1 cells proliferate at a slow rate in serum-free medium. This isenhanced by the addition of 7% FCS as shown in FIG. 7.

The addition of P-type PGK from mycobacteria causes a progressivedecrease in the proliferation of Y1 cells in the absence of serum (FIG.7). This result is the reverse of that seen with NIH-3T3 cellstransfected with the EGF receptor (see FIGS. 4 and 5).

The addition of A-type PGK from mycobacteria to Y1 cells in RPMI 1640without serum also causes inhibition of proliferation as shown in FIG.6, but this effect is maximal at an intermediate dilution, with lessinhibition when the PGK is very concentrated or very dilute. Thisdose/response curve is again the reverse of that seen when the same PGKpreparation is tested on the transfected NIH-3T3 cells (see FIGS. 2 and3).

The results are summarised in the following Table:

                  TABLE 1                                                         ______________________________________                                        Source of                                                                            PKA      PDH        Lipogensis                                         Second (%       (%         (%      EGF (Rc) 3T3                               Messenger                                                                            inhibition)                                                                            stimulation)                                                                             stimulation)                                                                          (growth)                                   ______________________________________                                        VB-P   53%      120%       --*     n.d.                                       VBS-P  n.d.     23%        --*     +                                          VSB-P  n.d.     42%        --*     +                                          VB-A   43%      12% (n.s.)  22%    n.d.                                       VBS-A  n.d.     --*         95%    +++                                        VSB-A  n.d.     --*        100%    +++                                        L-A    85%      --*        100%    +++                                        L-P    76%      38%        --*     +++                                        10% FCS                                                                              n.d.     n.d.       n.d.    +++                                        Insulin                                                                              n.d.     n.d.       273%    +                                          EGF    n.d.     n.d.       n.d.    +                                          ______________________________________                                         VBS-P: M. vaccaederived PGK, organism boiled then sonicated (mimics the       action of mammalian Ptype second messenger).                                  VSBP: M. vaccaederived PGK, organism sonicated then boiled (mimics the        action of mammalian Ptype second messenger).                                  VBSA: M. vaccaederived PGK, organism boiled then sonicated (mimics the        action of mammalian Atype second messenger).                                  VSBA: M. vaccaederived PGK, organism sonicated then boiled (mimics the        action of mammalian Atype second messenger).                                  VBP: M. vaccaederived PGK, organism boiled (mimics the action of mammalia     Ptype second messenger).                                                      VBA: M. vaccaederived PGK, organism boiled (mimics the action of mammalia     Atype second messenger).                                                      LA: Liver Atype IPG.                                                          LP: Liver Ptype IPG.                                                          NOTES:                                                                        *Atype is not active in PDH assay; Ptype is not active in lipogenesis         assay.                                                                        n.d. Not determined.                                                          +++ Proliferative data reported in FIGS. 1 to 5. All data were obtained i     the absence of 10% FCS unless indicated otherwise.                            n.s. Not significant.                                                    

For rat liver tissue, the material extraced from 16 g (wet weight) isdissolved in 0.2 ml of Hanks buffer (stock). Therefore, 10 μl of stockrepresents the amount of mediator recovered from 800 mg of startingtissue (wet weight).

For the lipogenesis assay, 10 μl of the stock solution is added to afinal volume of 1.25 ml.

For the PDH assay, 10 μl of the stock solution is added to a finalvolume of 0.27 ml.

For the PKA assay, 10 μl of the stock solution is added to a finalvolume of 0.1 ml.

For the cell proliferation assays, the dilutions quoted are finaldilutions. For example, 2.5 μl of the stock solution is added to a finalvolume of 0.1 ml, or 1/40 final dilution.

For M. vaccae, 10 μl of stock solution represents the amount of mediatorrecovered from 800 mg wet weight of bacteria. The amounts used in theassays are as described above for the rat liver tissues.

What is claimed is:
 1. A hormone or growth factor mimetic secondmessenger consisting essentially of at least one phosphoglycokineisolated from a microorganism of the genus Mycobacterium.
 2. A mimeticsecond messenger according to claim 1, which mimics the action ofinsulin, adrenocorticotrophic hormone (ACTH), nerve growth factor (NGF),epidermal growth factor (EGF), fibroblast growth factor (FGF),transforming growth factor β (TGFβ) or hepatocyte growth factor (HGF).3. A mimetic second messenger according to any of claim 1 wherein themycobacterium is Mycobacterium vaccae.
 4. A pharmaceutical compositioncomprising a hormone or growth factor mimetic second messenger accordingto claim
 1. 5. A method of treatment of type I or type II diabetesmellitus, polycystic ovary syndrome, lipodystrophy, age-related memoryloss and post-ischemic damage secondary to stroke or post transplantcomplications which method comprises administering an effective amountof an insulin mimetic second messenger consisting essentially of atleast one phosphoglycokine isolated from a microorganism of the genusMycobacterium to a patient in need thereof.
 6. A method of treatment ofnerve, spinal chord or central nervous system damage secondary totrauma, autoimmune or metabolic damage, or post ischemic damagesecondary to stroke or post-transplant complications, which methodcomprises administering an effective amount of a nerve or neurite growthfactor mimetic second messenger consisting essentially of at least onephosphoglycokine isolated from a microorganism of the genusMycobacterium to a patient in need thereof.
 7. A method of treatment ofhepatic damage caused by infection, alcohol abuse, drug sensitivity orautoimmunity, which method comprises administering an effective amountof a hepatocyte growth factor mimetic second messenger consistingessentially of at least one phosphoglycokine isolated from amicroorganism of the genus Mycobacterium to a patient in need thereof.8. A method of promotion of wound healing following surgery or trauma ortissue damage induced by ischemia or autoimmunity, which methodcomprises administering an effective amount of a fibroblast growthfactor mimetic second messenger consisting essentially of at least onephosphoglycokine and an epidermal growth factor mimetic second messengerisolated from a microorganism of the genus Mycobacterium to a patient inneed thereof.
 9. A method of treatment of a disease state involvingadrenal atrophy, which method comprises administering an effectiveamount of an adrenal cell growth factor mimetic second messengerconsisting essentially of at least one phosphoglycokine and an ACTHmimetic second messenger isolated from a microorganism of the genusMycobacterium to a patient in need thereof.
 10. The method according toclaim 9 wherein the disease state is tuberculosis.